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PubMed1999

Mouse-to-rabbit xenotransplantation: a new small animal model of hyperacute rejection mediated by the classical complement pathway.

BACKGROUND: Hyperacute rejection of porcine organs transplanted into primate recipients is initiated by the binding of preformed xenoreactive natural antibodies to the vascular endothelium of the graft and activation of the classical complement pathway. Several small animal models are currently employed to study various aspects of xenograft rejection; however, none has been shown to manifest hyperacute rejection mediated by the classical pathway of complement activation. METHODS: We performed heterotopic mouse heart transplants into weanling rabbits, adult rabbits, and C6-deficient rabbits. The recipients received no immunosuppression. Rejected grafts were subjected to histologic analysis and immunofluorescence staining for rabbit IgG, IgM, and C3. Levels of preexisting cytotoxic antibodies as well as classical and alternative complement pathway activities were determined in rabbit serum using mouse red cells as targets. RESULTS: Mean graft survival was 37+/-9.6 min for mouse-to-weanling rabbit transplants (n=10), and 40+/-11.1 min for mouse-to-adult rabbit transplants (n=5). Rejected grafts showed diffuse interstitial hemorrhage, endothelial cell damage, myocyte necrosis, moderate diffuse deposition of rabbit IgG, and dense deposition of rabbit IgM and C3 on the vascular endothelium of the graft, consistent with hyperacute rejection. One mouse-to-C6-deficient rabbit transplant was rejected at 21 hr with severe interstitial hemorrhage, cellular necrosis and a moderate cellular infiltrate consisting primarily of neutrophils and some mononuclear cells. A second transplant in a C6-deficient rabbit was functioning when the recipient died at 6.5 hr as a result of complications of surgery; the graft had normal myocytes and vasculature with minimal spotty interstitial hemorrhage. Both weanling and adult rabbit serum were found to have high titers of cytotoxic IgM anti-mouse antibodies and strong classical complement pathway activity with minimal alternative pathway activity towards mouse red cells. CONCLUSIONS: The mouse-to-rabbit species combination manifests hyperacute xenograft rejection. In vitro studies suggest that this process is mediated by IgM anti-mouse natural antibodies and activation of the classical pathway of complement.

Aging↗
PubMed2001

Intact B cell tolerance in the absence of the first component of the classical complement pathway.

A critical role for complement in the regulation of self tolerance has been proposed to explain the strong association between complement deficiency and autoimmunity. To elucidate the role of the classical pathway of complement in the maintenance of B cell tolerance, C1q-deficient (C1qa-/-) mice were bred with anti-hen egg lysozyme (HEL) immunoglobulin (Ig(HEL)) and soluble HEL (sHEL) transgenic mice. B cell tolerance was intact in C1qa-/- mice. In vivo, double-transgenic (Ig(HEL)/sHEL) C1qa-/- and wild-type control mice down-regulated surface immunoglobulin expression on splenocytes and equivalent numbers of HEL-binding B cells accumulated in the periphery. Maturation of B cells, evidenced by CD21 expression, was retarded to the same extent and at a similar time point. The frequency of anti-HEL-producing plasma cells and serum levels of anti-HEL immunoglobulin were comparably reduced in control and C1qa-/- double-transgenic mice compared to control Ig(HEL) and C1qa-/- Ig(HEL) mice. Furthermore, splenocytes from double-transgenic C1qa-/- or wild-type mice did not modulate intracellular calcium levels after stimulation with HEL in vitro. These data demonstrate that a stable form of B cell anergy persists in the periphery of C1qa-/- mice, suggesting that activation of the classical pathway by C1q is not essential for the maintenance of B cell tolerance in this transgenic model.

Animals↗
PubMed1984

Prevention of immune precipitation by purified classical pathway complement components.

The role of the classical pathway of complement in the prevention of immune precipitation has been investigated using purified complement components and immune complexes (IC) consisting of rabbit anti-BSA and BSA. C1 reduced the rate of immune precipitation. As C1q, EDTA treated C1 or C1-inhibitor treated C1 were unable to retard the precipitation of IC, it was concluded that the intact C1 molecule was required for this function. Use of phenylmethylsulphonyl fluoride and benzamidine showed that the enzymatic site on C1 was not required for this activity. C4 and C2 did not affect immune precipitation significantly when C1 was present at the concentrations present in serum. When C3 was added to C1, C4 and C2 precipitation of IC did not occur. These data demonstrate that classical pathway activation alone is sufficient for the prevention of immune precipitation.

Antigen-Antibody Complex↗
PubMed2007

Conformationally altered hyaluronan restricts complement classical pathway activation by binding to C1q, C1r, C1s, C2, C5 and C9, and suppresses WOX1 expression in prostate DU145 cells.

Linear non-sulfated hyaluronan (HA) does not bind complement proteins yet inhibits their hemolytic function. We have previously induced the complement inhibitory function of HA by heat treatment. However, heated HA readily loses its anti-complementary activity probably due to instantaneous interchain re-association. Here, HA solutions were heated and then freeze-dried. Compared to native HA, heated/freeze-dried HA stably restricted serum complement-mediated hemolysis via the classical pathway, in which serum C1 hemolytic function and C3 activation were blocked. Also, treated HA had a significantly increased binding of component C1q, C1r, C1s, C2, C5, C9, P, D and H. Further, when HA was gel-fractionated by electrophoresis and then freeze-dried, its anti-complementary activity was stably induced. Both native and heated/freeze-dried HA stimulated ERK phosphorylation in prostate DU145 cells. However, treated HA suppressed the expression of tumor suppressors WOX1 and WOX2. Together, HA with an altered conformation stabilizes its inhibition and binding of complement proteins. It may recognize cell surface receptors differently from native HA, thereby differentially regulating the expression of cellular proteins.

Cell Line, Tumor↗
PubMed1989

Inhibitory activity of Jatropha multifida latex on classical complement pathway activity in human serum mediated by a calcium-binding proanthocyanidin.

This study isolates and characterizes the anti-complement constituent(s) present in the latex of Jatropha multifida, in an attempt to explain the traditional application of the latex in the treatment of infected wounds. Guided by the inhibition of classical pathway (CP) complement activity in human serum, a polymer was isolated which could be characterized as a proanthocyanidin. The polymer inhibits CP activation of the complement cascade, while alternative pathway (AP) activation is relatively insensitive to the polymer. This is due to the selective depletion of Ca2+, but not Mg2+, from the incubation medium.

Anthocyanins↗
PubMed1979

Activation of the classical complement pathway by nephritic factor bound to the alternative pathway C3/C5 convertase.

Nephritic Factor (NF), the potent alternative pathway activator, which is occasionally found in association with certain types of nephritis has recently been identified as an IgG class autoantibody specific for the C3 convertase (C3bB) of the alternative pathway. In these studies we have examined the possibility that the cell-bound NF-stabilized C3 convertase (EC3 bBNF) binds and activates the first component of the classical pathway of complement. EC3bBNF bound C1q, and the extent of binding was dependent upon the number of NF molecules bound per cell and decreased parallel to the dissociation and release of NF from the cells. Interaction of C1 with bound NF resulted in its activation as shown by the proteolytic conversion of proenzyme C1s to its activated form C1s. As was the case with C1q binding, C1 activation was dependent on the number of NF molecules bound per cell. Thus the NF-stabilized C3 convertase binds and activates C1.

Binding Sites↗
PubMed2006

Classical pathway complement activity in Familial Mediterranean fever.

OBJECTIVES: The present study emphasizes the important role of the immune reactions in the pathogenesis of Familial Mediterranean fever. In the present study, the total hemolytic activity of the complement and the activities of individual complement components, C1, C2, C3, and C4, were determined in the blood serum of 32 patients with FMF and 28 healthy subjects. DESIGN AND METHODS: Hemolytic assay was applied, measuring THAC and individual complement components' activities. The patients were divided into 3 groups upon the regularity of colchicine therapy: patients receiving regular, irregular and not receiving colchicine treatment for at least 1 year. RESULTS: No significant changes in the hemolytic activities of the C1, C2, C3, and C4 complement components were found between the healthy subjects and those FMF patients, who were receiving regular colchicine treatment. CONCLUSIONS: Our data obtained have raised a number of important questions relevant to FMF pathogenesis and once again confirms the efficiency of regular colchicine treatment.

Adult↗
PubMed1985

C56 formation in the reaction mixture of isolated complement components through the classical complement pathway.

The mechanism of hemolysis of unsensitized erythrocytes by a mixture of 9 isolated, human-derived complement components, C1s, C4, C2, C3, C5, C6, C7, C8 and C9 (C1s-C9) was studied. Of the tested erythrocytes, guinea pig erythrocytes (Egp) were the most susceptible to lysis by C1s-C9, followed by human and sheep erythrocytes. Contamination of the isolated complement components by C56 was ruled out. It was determined that a factor was generated in the reaction mixture of C1s, C4, C2, C3, C5 and C6 (C1s-C6), which had lytic activity against Egp when C7, C8 and C9 were added. We found that the lytic factor was similar to C56 in the following properties: (1) the activity of the lytic factor decreased when incubated with isolated C7 prior to its reaction with Egp; (2) the lytic factor did not bind to Egp by itself but it did bind in the presence of C7; (3) EDTA did not have any inhibitory effect on the lytic factor; (4) the activity of the lytic factor decreased by treatment with anti-C5 and anti-C6 but not by treatment with anti-C3 and anti-C4, and (5) gel filtration of the reaction mixture (C1s-C6) indicated that the elution volumes of the lytic factor and of isolated C56 were similar. Thus, it is likely that C56 is generated in the reaction mixture of C1s-C6 and the lytic factor binds to unsensitized erythrocytes together with C7, to form an intermediate EC567 which is susceptible to lysis by the action of C8 and C9.

Animals↗
PubMed1987

[Components of the classical complement pathway in systemic lupus erythematosus].

Measurements of complement components in sera from patients with systemic lupus erythematosus (SLE) and some of their relatives indicated that decreased levels of CH50, C4 and C2 were mostly related to a genetic deficiency at one or both of the loci coding for C4, at least in those patients in whom decreased C4 levels were associated with normal C1 hemolytic activity. C4 deficiency is either isolated or associated with complement activation. In some patients with C4 deficiency, complement activation could only be demonstrated by measuring plasma level of the C3 cleavage fragment, C3a des Arg. Decreased concentration and/or hemolytic activity of C4 and C2 in SLE cannot be used to assess the activity of the disease.

Complement Activation↗
PubMed1996

Regulation of the B cell response to T-dependent antigens by classical pathway complement.

Mice deficient in complement components C3 (C3 -/-) and C4 (C4 -/-) were found to have a profound defect in their Ab response to a T-dependent Ag (bacteriophage (phi X174). Characterization of the deficient mice demonstrated a diminished level of peanut agglutinin+ germinal centers and a failure in isotype switching despite normal B cell signaling in vitro. The nature of the defect was found to lie at the B cell level, as the T cells were primed in C3- and C4-deficient mice as well as those in wild-type mice. These results, and the finding that the defect could be partly reversed by a 10-fold increase in Ag dose, support the hypothesis that covalent attachment of complement ligands, i.e., C3b and C3d to the Ag-Ab complex, increases its immunogenicity.

Animals↗
PubMed1996

Synthesis of classical pathway complement components by chondrocytes.

Using immunohistochemical studies, C1q, C1s, C4 and C2 were detected in chondrocytes in normal human articular cartilage and macroscopically normal articular cartilage from the inferior surfaces of hip joints of patients with osteoarthritis. Using reverse-transcribed polymerase chain reaction (RT-PCR), mRNA for C1q, C1s, C4 and C2 was also detected in RNA extracted from articular cartilage. C1r, C3, C1-inhibitor, C4-binding protein and factor I were not detected by either technique. Articular chondrocytes cultured in vitro synthesized C1r, C1s, C4, C2, C3 and C1-inhibitor but not C1q, C4-binding protein or factor I, as assessed by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis. Thus cultured articular chondrocytes have a complement profile that is similar to that of cultured human fibroblasts rather than that of articular chondrocytes in vivo. Complement synthesis in cultured chondrocytes was modulated by the cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), showing that cytokines can probably regulate complement synthesis in intact cartilage. The possible roles of local synthesis of complement components by chondrocytes in matrix turnover and the regulation chondrocyte function are discussed.

Base Sequence↗
PubMed2001

Continual low-level activation of the classical complement pathway.

There is evidence that the classical complement pathway may be activated via a "C1-tickover" mechanism, analogous to the C3-tickover of the alternative pathway. We have quantitated and characterized this pathway of complement activation. Analysis of freshly collected mouse and human plasma revealed that spontaneous C3 activation rapidly occurred with the generation of C3 fragments in the plasma. By the use of complement- and Ig-deficient mice it was found that C1q, C4, C2, and plasma Ig were all required for this spontaneous C3 activation, with the alternative complement pathway further amplifying C3 fragment generation. Study of plasma from a human with C1q deficiency before and after therapeutic C1q infusion confirmed the existence of a similar pathway for complement activation in humans. Elevated levels of plasma C3 were detected in mice deficient in complement components required for activation of either the classical or alternative complement pathways, supporting the hypothesis that there is continuous complement activation and C3 consumption through both these pathways in vivo. Blood stasis was found to stimulate C3 activation by classical pathway tick-over. This antigen-independent mechanism for classical pathway activation may augment activation of the complement system at sites of inflammation and infarction.

Animals↗
PubMed1995

Classical complement pathway activation on nucleated cells. Role of factor H in the control of deposited C3b.

The restriction of alternative complement pathway activation in fluid phase or on nonactivator surfaces has been described as the major physiologic function of the complement regulatory protein factor H. In this study, we provide evidence that factor H is also a restriction factor of classical pathway activation on the surface of nucleated cells. We found that C3b was rapidly converted to inactivated C3b (iC3b) on human SK-MEL-93-2 melanoma cells after classical pathway activation with the murine monoclonal IgG3 Ab R24 directed against the disialoganglioside surface Ag GD3. The SK-MEL-93-2 cells are nonactivators of the alternative pathway and express neither CR1 (CD35) nor the C3b-cleaving protease p65. The cells are further characterized by the expression of only moderate amounts of DAF (CD55) and approximately 5 x 10(3) MCP (CD46) molecules/cell. FACS analysis and direct quantitation using [125I]factor H revealed high level binding of factor H to the melanoma cells (5.6 x 10(6) molecules/cell) during classical pathway activation. The binding of factor H could be inhibited under conditions that inactivate the classical complement pathway (EGTA and heat treatment), but not by factor B depletion of the serum, demonstrating that classical pathway activation was responsible for factor H binding. Treatment of factor B-depleted serum with neutralizing concentrations of polyclonal anti-factor H resulted in the prolonged presence of intact C3b on the cells and a significantly reduced generation of iC3b. The increased amount of C3b on these cells correlated with a 2.65-fold greater rate of cell death. In contrast, the increase in cell death effected by neutralizing concentrations of anti-CD46 or anti-CD55 Ab was only 0.13- or 0.35-fold, respectively. In addition, the supplementation of serum with purified factor H decreased the extent of lysis of the cells. Collectively, these data provide experimental evidence that factor H, through its cofactor activity for C3b degradation, is involved in the restriction of the classical pathway of complement on the surface of nucleated cells, a function that to date has been exclusively attributed to the membrane regulatory proteins CD35 and CD46.

Complement Activation↗
PubMed2006

Complement regulation in human atherosclerotic coronary lesions. Immunohistochemical evidence that C4b-binding protein negatively regulates the classical complement pathway, and that C5b-9 is formed via the alternative complement pathway.

OBJECTIVE: The complement system is activated in human atherosclerotic lesions and may hence aggravate local inflammation. We studied the presence and localization of C4b-binding protein (C4bp), the major inhibitor of the classical complement pathway, in human atherosclerotic lesions in relation to complement activation products and protein S, which circulates in complex with C4bp. METHODS AND RESULTS: Immunohistochemistry of human coronary arteries showed C4bp to be virtually absent in normal arteries but present in early and advanced atherosclerotic lesions. In the lesions, C4bp is associated with proteoglycans, and affinity chromatography showed that C4bp interacts with human arterial proteoglycans. Areas containing C4bp also contained IgM and C4 suggesting that C4bp is involved in the regulation of the classical complement pathway. However, C5b-9 was virtually absent in these areas but, instead, colocalized with properdin deeper in the intima, suggesting that C5b-9 is formed by the alternative complement pathway. A fraction of C4bp was associated with protein S and apoptotic cells. CONCLUSIONS: The results indicate that C4bp regulates the classical complement pathway in human atherosclerotic lesions. Thus, unlike the alternative pathway, the classical complement pathway does not generate C5b-9, but is likely to be involved in the clean-up of apoptotic cells and cell debris in the arterial intima.

Apoptosis↗
PubMed1988

Epstein-Barr virus transformed B cell lines derived from patients with systemic lupus erythematosus produce a nephritic factor of the classical complement pathway.

Nephritic factor of the classical complement pathway (C4NeF) is an IgG antibody which stabilizes the C3 convertase (C4b2a) and has been detected in sera from patients with systemic lupus erythematosus (SLE) and acute postinfectious glomerulonephritis. In order to study the production of nephritic factor (NeF), mononuclear cells were isolated from the peripheral blood of patients with SLE and infected with Epstein-Barr virus (EBV) to establish active B lymphocyte cell lines. Supernatants from 15 established B cell lines, as well as from 10 B cell lines established from normal individuals, were investigated for their ability to conserve the classical and the alternative pathway C3 convertases as assessed by EAC3bBb and EAC14b2a stabilizing assays. Supernatants from 2 of 15 B cell lines from patients with SLE, but none from normal individuals, stabilized the classical C3 convertase without having any effect on the alternative pathway C3 convertase. Using anti-human Ig affinity chromatography, we showed that C4NeF activity resided in the IgG fraction; the IgG fraction containing C4NeF activity bound to the C4b2a complex, but not to C4b alone. On gel electrophoresis, following reduction, the heavy chains were slightly heavier than the heavy chains of normal IgG. We were able to isolate C4NeF from the sera of the 2 patients with SLE from whom the positive supernatants were derived, but were unable to detect any C4NeF activity in the sera of the other 13 patients and the 10 normal individuals. Serum and B cell line supernatant-derived C4NeF exhibited comparable characteristics. We conclude that C4NeF produced in vitro by EBV-transformed B cell lines derived from patients with SLE is functionally similar to the conventional C4NeF in serum. These studies confirm the production of autoantibodies by B cells with the ability to stabilize the classical pathway C3 convertase in certain patients with SLE; stabilization of the C4b2a enzyme in these patients is an apparent mechanism for the development of hypocomplementemia. Finally, preparation of homogeneous C4NeF in vitro should improve our understanding of the role of autoantibodies in complement metabolic disturbances in autoimmune diseases.

Autoantibodies↗
PubMed1987

Covalent association of C3b with C4b within C5 convertase of the classical complement pathway.

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

Animals↗
PubMed2003

Importance of the prime subsites of the C1s protease of the classical complement pathway for recognition of substrates.

The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P(1)'-P(4)' substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When C1s was in complex with C1q and C1r, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C1s for prime side amino acids by examining a substrate library in which each of the P(1)'-P(4)' positions had been substituted by different classes of amino acids. This revealed that the P(1)' position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P(1)'-P(4)' positions abolished the allosteric behavior, indicating that contact residues at these positions in the C1s enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates.

Amino Acid Substitution↗